Use of Derivatives of Polyunsaturated Fatty Acids as Medicaments

ABSTRACT

Use of polyunsaturated fatty acid derivatives as medicaments or functional foods. The present invention relates to the use of 1,2-fatty acid derivatives in the treatment or prevention of common diseases whose etiology is based on alterations (of any type) of the cell membrane lipids, for example, changes in levels, in the composition or in the structure of these lipids. In addition, for diseases in which the regulation of lipid composition and of the structure of the membranes (or proteins that interact with membranes) causes the reversion of pathological state.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/683,379, which is a divisional of U.S. application Ser. No.14/869,080, which is a continuation of U.S. application Ser. No.13/257,128, which is the National Stage of International Application No.PCT/ES2010/070153, filed Mar. 15, 2010, which claims foreign prioritybenefit from Provisional Spanish Patent Application No. P200900725,filed on Mar. 16, 2009, all of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the use of 1,2-polyunsaturated fattyacid derivatives as medicaments, preferably for the treatment ofdiseases whose etiology is based on alterations of cell membrane lipids,such as: changes in the levels, in the composition or structure of theselipids and proteins that interact with them; as well as in the treatmentof diseases where the regulation of lipid composition and membranestructure, as well as of proteins that interact with them with theresult of reversion a of pathological state.

Thus, the present invention, because of its wide range of application,is likely to be generally included in the field of medicine andpharmacy.

STATE OF THE ART

Cell membranes are structures that define the organization of cells andthe organelles they contain. Most biological processes occur in oraround membranes. Lipids not only have a structural role, but alsoregulate the activity of important processes. Moreover, the regulationof the membrane lipid composition also influences the location orfunction of important proteins involved in controlling the cell'sphysiology, such as G proteins or PKC (Escribá et al., 1995, 1997, Yanget al, 2005, Martinez et al., 2005). These and other studies demonstratethe importance of lipids in controlling important cellular functions. Infact, many human diseases such as cancer, cardiovascular disease,neurodegenerative diseases, obesity, metabolic disorders, processes andinflammatory diseases, infectious diseases or autoimmune diseases, amongothers, have been associated with alterations in the levels or thecomposition of lipids in biological membranes, further demonstrating thebeneficial effects that treatments with fatty acids could be used toreverse these diseases, in addition to those of the present invention,which regulate the composition and structure of membrane lipids(Escribá, 2006).

The lipids consumed in the diet regulate the lipid composition of cellmembranes (Alemany et al., 2007). In addition, various physiological andpathological situations can change lipids in cell membranes (Buda etal., 1994; Escribá, 2006). As an example of a situation that inducesphysiological changes in membrane lipids it may be mentioned the fishliving in rivers with variable temperature, whose lipids undergoimportant changes (changes in the quantity and types of membrane lipids)when the temperature goes down from 20° C. (summer) to 4° C. (winter)(Buda et al. 1994). These changes allow the maintenance of theirfunctions in cell types of diverse nature. Examples of pathologicalprocesses that may influence the lipid composition are neurologicaldisorders or drug-induced diseases (Rapoport, 2008). Therefore, onecould say that membrane lipids can determine correct activity ofmultiple mechanisms of cell signalling.

Changes in membrane lipid composition affect cell signalling and maylead to development of disease or to reverse them (Escribá, 2006).Various studies over the past few years indicate that membrane lipidsplay a more relevant role than they had been assigned so far (Escribá etal., 2008). The classical view of the cell membrane assigns to lipids apurely structural role, as a support for membrane proteins, which aresupposed to be the only functional elements of the membrane. The plasmamembrane would have an additional role, avoiding water, ions and othermolecules from entering into the cells. However, membranes have otherfunctions of great importance in the maintenance of health, diseaseoccurrence and healing. Since the body is sick because their cells aresick, alterations in membrane lipids produce alterations in cells andthese can lead to the occurrence of diseases. Similarly, therapeutic,nutraceutical or cosmetic interventions, aimed at the regulation of thelevels of membrane lipids can prevent and reverse (cure) pathologicalprocesses. In addition, numerous studies indicate that consumption ofsaturated and trans-monounsaturated fats is related to the deteriorationof health. In addition to the neurological diseases described above,vascular diseases, cancer and others have also been directly associatedwith membrane lipids (Stender and Dyerberg, 2004). The deterioration ofan organism is manifested in the appearance of this and other types ofdiseases, which may include metabolic diseases, inflammation,neurodegeneration, etc.

Cell membranes are the selective barrier through which a cell receivesmetabolites and information from other cells and the extracellularenvironment that surrounds it. However, membranes develop other veryimport functions in cells. On the one hand, they serve as a support forproteins involved in receiving or initiating messages that controlimportant organic functions. These messages, which are mediated by manyhormones, neurotransmitters, cytokines, growth factors, etc., doactivate membrane proteins (receptors), which propagate the receivedsignal into the cell through other proteins (peripheral membraneproteins), some of which are also located at the membrane. Since (1)these systems work as amplification cascades, and (2) membrane lipidscan regulate the localization and activity of these peripheral proteins,the lipid composition of membranes can have a major impact on cell'sphysiology. In particular, the interaction of certain peripheralproteins, such as G proteins, protein kinase C, Ras protein, etc., withthe cell membrane depends on its lipid composition (Vogler et al., 2004,Vogler et al., 2008). Furthermore, the lipid composition of cellmembranes is influenced by the type and amount of lipids in the diet(Escribá et al., 2003). In fact, nutraceutical or pharmaceutical lipidinterventions can regulate the lipid composition of membranes, which inturn can control the interaction (and hence the activities) of importantcell signalling proteins (Yang et al., 2005).

The fact that membrane lipids are able to control cell signalling, mayalso suppose that they are able to regulate the physiological status ofcells and therefore the general state of health. In fact, both negativeand positive effects of lipids on health have been described (Escribá etal., 2006; Escribá et al., 2008). Preliminary studies have shown that2-hydroxyoleic acid, a monounsaturated fatty acid, is able to reversecertain pathological processes such as overweight, hypertension orcancer (Alemany et al., 2004, Martinez et al., 2005; Vogler et al,2008).

Cardiovascular diseases are often associated with excessiveproliferation of cells that constitute the heart and vascular tissues.This hyperproliferation results in cardiovascular deposits in the innerlumen of vessels and cavities of the cardiovascular system resulting ina wide range of diseases such as hypertension, atherosclerosis,ischemia, aneurysms, ictus, infarction, angina, stroke (cerebrovascularaccidents) etc. (Schwartz et al., 1986). In fact, it has been suggestedthat the development of drugs that prevent cell proliferation would be agood alternative for prevention and treatment of cardiovascular disease(Jackson and Schwartz, 1992).

Obesity is caused by an altered balance between intake and energyexpenditure, in part due to alterations in the mechanisms regulatingthese processes. On the other hand, this condition is characterized byhyperplasia (increase in cell number) or hypertrophy (increased size) offat cells, adipocytes. Numerous studies show that fatty acids eitherfree or as part of other molecules, may influence a number of parametersrelated to energy homeostasis, such as body fat mass, lipid metabolism,thermogenesis and food intake, among others (Vogler et al., 2008). Inthis sense, the modification of fatty acids could be a strategy toregulate energy homeostasis, i.e., the balance between intake and energyexpenditure, and therefore related processes such as appetite or bodyweight.

Neurodegenerative processes lead to a number of diseases with differentmanifestations, but with the common characteristic of being caused bydegeneration or dysfunction of the central and/or peripheral nervoussystem cells. Some of these neurodegenerative processes involve asignificant reduction in the cognitive ability of patients oralterations of their motor ability. Neurodegenerative, neurological andneuropsychiatric disorders have a common basis of neuronal degenerationor alteration of its components, such as lipids (e.g., myelin) ormembrane proteins (e.g., adrenergic, serotonergic receptors, etc.). Suchcentral nervous system diseases include, among others, Alzheimer'sdisease, Parkinson's disease, Multiple sclerosis, ALS, sclerosis of thehippocampus and other types of epilepsy, focal sclerosis,adrenoleukodystrophy and other leukodystrophy, vascular dementia, seniledementia, headaches including migraine, central nervous system trauma,sleep disorders, dizziness, pain, stroke (cerebrovascular accidents),depression, anxiety, or addictions. Furthermore, certain neurologicaland neurodegenerative diseases may lead to processes that end up inblindness, hearing problems, disorientation, altered mood, etc.

An example of well-characterized neurodegenerative disorder isAlzheimer's disease, characterized by the formation of senile plaques,composed of membrane protein fragments (eg β-amiloyd peptide) originatedfrom a wrong peptide processing, followed by an accumulation on theoutside of the cells, and neurofibrillary tangles of Tau protein. Thisprocess has been associated with alterations in the metabolism ofcholesterol and the consequent alteration of the levels of certainmembrane lipids such as cholesterol and docosahexaenoic acid (Sagin andSozmen, 2008, Rapoport, 2008). In addition, several neurodegenerativediseases such as Parkinson's disease, Alzheimer's disease, seniledementia (or Lewy bodies) have been associated with pathologicalaccumulation of fibrillar aggregates of the α-synuclein protein, whichlead to a significant alteration of the cellular metabolism oftriglycerides (Coles et al., 2001). In fact, the development of theseand other neurodegenerative diseases is associated with alterations inserum or cell lipids, such as cholesterol, triglycerides, sphingomyelin,phosphatidylethanolamine, etc. This again suggests that lipids play acrucial role in the correct activity of neurons, nerves, brain,cerebellum and spinal cord, which is logical given the abundance oflipids in the central nervous system. The molecules of this inventionhave a high or very high potential to reverse many of the processesassociated with neurological, neurodegenerative and neuropsychiatricdisorders.

Moreover, different types of sclerosis and other neurodegenerativediseases related to the “demyelination”, whose net result is the loss oflipids on the cover of the neuronal axons, with consequent changes inthe process of propagation of electrical signals that this involves.Myelin is a fatty layer that surrounds the axons of many neurons andthat is formed by a series of spiral folds of the plasma membrane ofglial cells (Schwann cells). Therefore, it's clear that lipids play animportant role in the development of neurodegenerative diseases.Moreover, it was found that unmodified natural PUFAs have a moderatepreventive effect on the development of neurodegenerative processes(Lane and Farlow, 2005). In fact, the most important lipid in thecentral nervous system is docosahexaenoic acid, a natural PUFA and whoseabundance is altered in many neurodegenerative processes.

Metabolic diseases form a group of diseases characterized by theaccumulation or deficit of certain molecules. A typical example isaccumulation of glucose, cholesterol and/or triglycerides above normallevels. The increased levels of glucose, cholesterol and/ortriglycerides, both systemic (e.g., increased plasma levels) and atcellular level (e.g., in cell membranes) is associated with alterationsin cell signalling leading to dysfunction at various levels, and areusually due to errors in the activity of certain enzymes or to theinadequate control of such proteins. Among the most important metabolicdisease are hypercholesterolemia (high cholesterol) andhypertriglyceridemia (high triglycerides). These diseases have higherrates of incidence, morbidity and mortality, so their treatment is anecessity of first order. Other important metabolic diseases includediabetes and insulin resistance, characterized by problems in thecontrol of glucose levels. These metabolic diseases are involved in theoccurrence of other diseases, like cancer, hypertension, obesity,atherosclerosis, etc. Recently, it has been defined another diseaseprocess closely related to metabolic disorders described above and whichcould constitute a new type of metabolopathy per se, it is the metabolicsyndrome.

The protective role of certain polyunsaturated fatty acids (PUFAs) oncertain diseases has been described by different researchers. Forexample, PUFAs slow the development of cancer and have positive effectsagainst the development of cardiovascular disease, neurodegenerativediseases, metabolic disorders, obesity, inflammation, etc. (Trombetta etal., 2007, Jung et al., 2008, Florent et al., 2006). These stimuliindicate the important role of lipids (PUFA) in both the etiology ofvarious diseases and in its treatment. However, the pharmacologicalactivity of these compounds (PUFA) is very limited due to rapidmetabolism and short half-life in blood. Therefore it seems necessary todevelop PUFAs with a slower metabolism, which results in an increasedpresence in the cell membrane compared to the PUFAs used up to now,facilitating the interaction of cell signalling peripheral proteins. Themolecules of this invention are synthetic derivatives of PUFAs, have aslower metabolism and a marked and significantly superior therapeuticeffect compared to the natural PUFAs.

Because of the relationship between structural and functionalalterations of lipids located in the cell membrane with the developmentof various diseases of different typology, but with an etiologyunitarily related to structural and/or functional alteration of lipidsin membrane cells, such as cancer, cardiovascular disease, obesity,inflammation, neurodegenerative and metabolic diseases, the presentinvention focuses on the use of new synthetic polyunsaturated fattyacids able to solve the technical problems associated with known fattyacids mentioned above and therefore, they are useful for treating thesediseases effectively.

DESCRIPTION OF THE INVENTION Brief Description of the Invention

This invention is focused on 1,2-derivatives of polyunsaturated fattyacids (hereinafter: D-PUFAs) for use in the treatment of common diseaseswhose etiology is related to structural and/or functional alterations ofcell membrane lipids, or of the proteins that interact with them,particularly selected from: cancer, vascular diseases, neurodegenerativeand neurological disorders, metabolic diseases, inflammatory diseases,obesity and overweight. D-PUFAs have a lower metabolic rate than naturalpolyunsaturated fatty acids (hereinafter: PUFA), because the presence ofdifferent atoms other than hydrogen (H) at carbons 1 and/or 2 blocks itsdegradation through β-oxidation. This causes significant changes in thecomposition of membranes, regulating the interaction of cell signallingperipheral proteins. This may lead to, for example, differences in thepackaging of the surface of the membrane, modulating the anchoring ofperipheral proteins that participate in the propagation of cellularmessages. Thus, the D-PUFA molecules that are the subject of thisinvention have an activity much greater than the PUFAs, showingsignificantly higher effect for the pharmacological treatment of theidentified diseases.

As mentioned above, the diseases treated with the D-PUFA molecules ofthe invention share the same etiology, which is related to structuraland/or functional (or any other origin) alterations of cell membranelipids or of the proteins that interact with them. The followingdiseases are listed as an example:

-   -   Cancer: liver cancer, breast cancer, leukaemia, brain cancer,        lung cancer, etc.    -   Vascular diseases: atherosclerosis, ischemia, aneurysms, ictus,        cardiomyopathy, angiogenesis, cardiac hyperplasia, hypertension,        infarction, angina, stroke (cerebrovascular accident), etc.    -   Obesity, overweight, appetite control and cellulite.    -   Metabolic diseases: hypercholesterolemia, hypertriglyceridemia,        diabetes, insulin resistance, etc.    -   Neurodegenerative diseases, neurological and neuropsychiatric        disorders: Alzheimer's disease, vascular dementia, Zellweger        syndrome, Parkinson's disease, multiple sclerosis, amyotrophic        lateral sclerosis, hippocampal sclerosis and other types of        epilepsy, focal sclerosis, adrenoleukodystrophy and other types        of leukodystrophy, vascular dementia, senile dementia, dementia        of Lewy, multiple systemic atrophy, prion diseases, headaches        including migraine, central nervous system injury, sleep        disorders, dizziness, pain, stroke (cerebrovascular accidents),        depression, anxiety, addictions, memory, learning or cognitive        problems and general diseases requiring stop of        neurodegeneration or neuroregeneration induced by the treatment        with the compounds of the invention.    -   Inflammatory diseases, including inflammation, cardiovascular        inflammation, tumour induced inflammation, inflammation of        rheumatoid origin, inflammation of infectious origin,        respiratory inflammation, acute and chronic inflammation,        inflammatory nature hyperalgesia, edema, inflammation resulting        from trauma or burns, etc.

The D-PUFA compounds of the present invention are characterized by thefollowing formula (I):

COOR₁—CHR₂—(CH₂)_(a)—(CH═CH—CH₂)_(b)—(CH₂)_(c)—CH₃   (I)

where a, b and c can have independent values between 0 and 7, and R₁ andR₂ may be an ion, atom or group of atoms with a molecular weight thatindependently do not exceed 200 Da.

In a preferred structure of the invention a, b and c can haveindependent values between 0 and 7, R₁ is H and R₂ is OH.

In another preferred structure of the invention a, b and c can haveindependent values between 0 and 7, R₁ is Na and R₂ is OH.

In another preferred structure of the invention a and c can haveindependent values between 0 and 7, b can have independent valuesbetween 2 and 7, and R₁ and R₂ may be an ion, atom or group of atomswhose molecular weight is independently equal or less to 200 Da.

The administration of the fatty acids of the invention can be carriedout by any means, for example enterally (IP), orally, rectally,topically, by inhalation or by intravenous, intramuscular orsubcutaneous injection. In addition, the administration may be eitheraccording to the formula above or in any pharmaceutically acceptablederivative from it, such as: esters, ethers, alkyl, acyl, phosphate,sulfate, ethyl, methyl, propyl, salts, complexes, etc.

In addition the fatty acids of the invention can be administered aloneor formulated in pharmaceutical or nutraceutical compositions whichcombine with each other and/or with excipients s such as: binders,fillers, disintegrators, lubricants, coaters, sweeteners, flavouringexcipients, colouring excipients, transporters, etc. and combinations ofall of them. Also, the fatty acids of the invention can be part ofpharmaceutical or nutraceutical compositions in combination with otheractive ingredients.

For the purposes of the present invention the term “nutraceutical” isdefined as a compound that is ingested regularly during feeding and actsto prevent diseases, in this case, with an etiology linked toalterations of cell membrane lipids.

For the purposes of the present invention the term “therapeuticallyeffective amount” is one that reverses or prevents the disease withoutshowing adverse side effects.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of compounds in Table 1 on tumour cell growth.On the y axis it is represented the number of viable cells (% control)depending on the compound used (x-axis). Human lung cancer (A549) cellswere cultured in RPMI-1640 with 10% serum for 48 hours in the absence(control) or presence of 250 μM of the compounds of the invention. Thegraph represents the number of viable cells (mean and standard error ofthe mean of three experiments). The dotted line represents the totalelimination of cells (0% viability).

FIG. 2 shows the effect of certain PUFAs and D-PUFA molecules of thepresent invention on the proliferation of A10 vascular cells. On the yaxis it is represented the number of cells (% control) depending on thefatty acid used (horizontal axis). The cells were incubated in completemedium (control, C), incomplete medium without supplement (CSS) orcomplete medium in the presence of PUFAs (182, 183A, 183G, 204, 205 and226) or D-PUFAs (182A1, 183A1, 183A2, 204A1, 205A1 and 226A1). Thereduction of proliferation, but still above the values of CSS, indicatesthat these molecules have the capacity to regulate abnormalproliferation of cardiovascular cells without being toxic.

FIG. 3A shows proliferation of adipocytes cultured in the absence(control, C) or presence of different D-PUFAs and PUFAs. On the y axisit is represented the number of cells (% control) depending on the fattyacid used (x axis). As non-proliferation control, a serum deficientmedium (medium with low serum percentage, MSB) was used. FIG. 3B showsin the Y-axis the body weight (% of untreated control) and thehorizontal axis the compounds used in the treatment of experimentalanimals. In the X axis, from left to right, it is represented first thetreatment with vehicle (C) and then, the treatment with several of thecompounds of the invention. SHR rats were treated for one month with 200mg/kg of each one or of the 24 compounds shown in the Figure. Eachexperimental group consisted of six animals and for each series a groupof animals treated with vehicle (water) was used, and results werecompared with the weight of the animals that had not received anytreatment. The letters A, B, N and P indicate the combination ofradicals R₁ and R₂ according to Table 3.

FIG. 4A shows death of P19 cells cultured in the absence of externalfactors (control, C: 0% neuronal death) and in the presence of NMDA(100% neuronal death). On the vertical axis it is represented theneuronal death (% of control) depending on the fatty acid used (x-axis).The presence of PUFAs induced modest increases in the survival of P19cells in the presence of NMDA. D-PUFAs induced significant increases incell survival values, exceeding in more than 200% in the case of 226A1.Since the number of cells in cultures treated cells is higher than incontrol cells, it may be affirmed that these compounds not only preventneuronal death induced by NMDA (anti-neurodegenerative) but also areneuroregenerative agents. FIG. 4B shows the effect of D-226B1 PUFA inimproving exercise performance in the radial maze in an animal model ofAlzheimer's disease. In the Y axis of the left figure it is shown thetime taken to complete the exercise and in the vertical Y axis of theright figure the total number of errors made in the implementation ofprogrammed exercise (mean±standard error of the mean) (runtime). In bothfigures, from left to right, it is represented in the X axis the resultsin healthy mice (control) (first column), in mice with induced Alzheimerand treated with water as vehicle (second column) or in mice treatedwith the compound 226B1 (third column). Animals with Alzheimer's diseasetook longer and made more errors than healthy mice, being thedifferences statistically significant (*, P<0.05). By contrast, micewith Alzheimer that were treated with the compound 226 B1 showed nosignificant differences with healthy animals.

FIG. 5A presents an immunoblot that shows the inhibition of theexpression of the pro-inflammatory COX-2 protein, induced previously bybacterial lipopolysaccharide (LPS) (C+, 100%) in human macrophagesderived from monocytes U937 by different D− PUFA of the presentinvention. FIG. 5A also presents a drawing showing the COX-2/COX-1relationship as % of control (Y axis) for the following compounds(X-axis): OOA (2-hydroxy-oleic acid), OLA (182A1), OALA (183A1), OGLA(183A2), OARA (204A1), OEPA (205A1), ODHA (226A1). FIG. 5B shows theanti-inflammatory efficacy of different D-PUFA compounds of the presentinvention in an animal model of inflammation. It shows the inhibitoryeffect on serum levels of TNFα (pg/ml) induced by LPS in mice (y axis)for different compounds of the invention (X axis). The reduction of thisfactor is directly related to the anti-inflammatory medication. Thecompounds are the same as in the left pane.

FIG. 6A shows Cholesterol levels in 3T3-L1 cells. On the vertical axisit is represented the levels of cholesterol (% total lipids) dependingon the fatty acid used (x-axis).

FIG. 6B shows total triglycerides in 3T3-L1 cells. On the vertical axisit is represented the levels triglycerides (% total lipids) depending onthe fatty acid used (x-axis). Shown values are mean±standard error ofthe mean of cholesterol and triglycerides compared to the total lipidsin cell membranes measured by spectrophotometric methods (cholesterol)or thin layer chromatography followed by gas chromatography(triglycerides). The graphs show the quantified values in cells culturedin the absence (Control) or presence of the D-PUFAs or PUFAs listedabove.

FIG. 7A shows the relationship between the structure of membrane andcellular effects induced by D-PUFAs. It is represented in the ordinateaxis the cellular effects (% control) compared to H_(II) transitiontemperature (X-axis). The mean of the effect of each of the D-PUFAmolecules was determined (average effect of each lipid in all diseasemodels studied and the number of double bonds) and it is plotted againstthe transition temperature. The reduction in H_(II) transitiontemperature indicates a greater induction of membrane discontinuities,which results in the presence of anchoring sites in the membrane forperipheral proteins and leads to better regulation of cell signallingand, therefore, more effective for the control of certain diseases.

FIG. 7B shows the relationship between therapeutic efficacy of PUFAs(empty circles) and D-PUFAs (solid circles.) Each point is the averageof the effect observed for all diseases studied (Y axis: change withrespect to control %) depending on the number of double bonds presentedby each molecule (horizontal axis). In both cases the correlations weresignificant (P<0.05). It was observed that the therapeutic effectdepends on the number of double bonds that the molecule has, which inturn is related to the ability to regulate the membrane structure. Inthis sense, the presence of a radical in carbons 1 and 2, present inD-PUFAs, but not in PUFAs, is essential to enhance the therapeuticeffect of these molecules.

These results indicate that the effects of lipids contained in thisinvention have a common basis. These correlations (with r² values of0.77 and 0.9 for D-PUFAs and P<0.05 in both cases) clearly indicate thatthe structure of the lipids used is the basis of its effect and that itoccurs through the regulation of membrane structure, caused by thestructure-function relationship of each lipid. In fact, there is anumber of research works in which human diseases are associated withalterations described above in the levels of PUFAs, demonstrating theimportant role of lipids in cellular physiology.

DETAILED DESCRIPTION OF THE INVENTION

The broad spectrum of therapeutic applications offered by D-PUFAmolecules of the present invention leads to widely assume that theseD-PUFA molecules confer the membranes with specific structuralproperties that allow the proper processing of the activity carried outin and through these membranes. In other words, many of theabnormalities that give rise to different kinds of diseases are causedby significant variations in the levels of certain important lipids forcell function and/or of proteins that interact with membranes and/or arerelated to production of lipids. These pathological changes that maylead to different kinds of diseases can be prevented or reversed bysynthetic fatty acids described in this invention, which can beeffectively used to treat or prevent any disease whose etiology isrelated either to alterations in levels, composition, structure, or anyother alteration of the biological membrane lipids or with aderegulation of cell signalling as a result of these changes in theselipids in biological membranes. Additionally, the lipids contained inthis invention can also be used as medicines when a disease occurs as aresult of another change, as long as the result of modulation of theproperties and/or membrane functions is able to reverse the pathologicalprocess.

For this study of the therapeutic effects of the fatty acids of thisinvention, cultured cell lines and animal models of various diseaseswere used and the activity of D-PUFAs and PUFAs to treat differentdiseases was investigated.

The structure of the molecules of the invention is shown in the Tables1, 2 and 3. Given the Formula I, compounds of the present inventionpreferably present combinations of the values of a, b and c shown inTable 1.

In addition, in the invention the compounds are named with a three digitnumber followed by the symbol X1 or X2. The number 1 denotes all D-PUFAsused, except the series based on C18:3 ω-6 (γ-linolenic acid), whichappear under number 2. The first two digits of this number represent thenumber of carbons of the molecule. The third digit of that numberrepresents the number of double bonds. The letter X is replaced by anyof the letters from A to W (Table 3), these letters A to W to representthe specific combination of R₁ and R₂ of Formula I.

Thus, particularly preferred compound of this invention are identifiedunder abbreviations: 182X1, 183X1, 183X2, 204X1, 205X1, 226X1 and shouldbe interpreted according to the above directions.

TABLE 1 D-PUFA a b c 182X1 Series 6 2 3 183X1 Series 6 3 0 183X2 Series3 3 3 204X1 Series 2 4 3 205X1 Series 2 5 0 226X1 Series 2 6 0

Table 2 shows the structures of some of the D-PUFA molecules of theinvention and the PUFAs from which they derive. As can be seen thattable illustrates some compounds of the invention with differentcombinations of values of a, b and c, and where the radicals R₁ and R₂are marked with the letter A, which means, as described above, that R₁is H and R₂ is OH (see Table 3).

TABLE 2 Name of the molecule Structure Prop Abbr.2-hydroxy-9,12-octadecadienoic acidCOOH—CHOH—(CH₂)₆—(CH═CH—CH₂)₂—(CH₂)₃—CH₃ S, OH 182A12-hydroxy-9,12,15-octadecatrienoic acid COOH—CHOH—(CH₂)₆—(CH═CH—CH₂)—CH₃S, OH 183A1 2-hydroxy-6,9,12-octadecatrienoic acidCOOH—CHOH—(CH₂)₃—(CH═CH—CH₂)₃—(CH₂)₃—CH₃ S, OH 183A22-hydroxy-5,8,11,14-eicosatetraenoic acidCOOH—CHOH—(CH₂)₂—(CH═CH—CH₂)₄—(CH₂)₃—CH₃ S, OH 204A12-hydroxy-5,8,11,14,17-eicosapentaenoic acidCOOH—CHOH—(CH₂)₂—(CH═CH—CH₂)₅—CH₃ S, OH 205A12-hydroxy-4,8,11,14,17-docosahexaenoic acidCOOH—CHOH—CH₂—(CH═CH—CH₂)₆—CH₃ S, OH 226A1 9,12-octadecadienoic acidCOOH—(CH₂)₇—(CH═CH—CH₂)₂—(CH₂)₃—CH₃ N 182 9,12,15-octadecatrienoic acidCOOH—(CH₂)₇—(CH═CH—CH₂)₃—CH₃ N 183A 6,9,12-octadecatrienoic acidCOOH—(CH₂)₄—(CH═CH—CH₂)₃—(CH₂)₃—CH₃ N 183G 5,8,11,14-eicosatetraenoicacid COOH—(CH₂)₃—(CH═CH—CH₂)₄—(CH₂)₃—CH₃ N 2045,8,11,14,17-eicosapentaenoic acid COOH—(CH₂)₃—(CH═CH—CH₂)₅—CH₃ N 2054,7,10,13,16,19-docosahexaenoic acid COOH—(CH₂)₂—(CH═CH—CH₂)₆—CH₃ N 226Prop: property. S: synthetic. N: natural. OH: hydroxylated on carbon 2(α carbon).

Table 3 shows the different combinations of radicals R₁ and R₂ that canbe combined with the values of a, b and c listed in Table 1.

TABLE 3 R₁ R₂ H Na K CH₃O CH₃—CH₂O OPO(O—CH₂—CH₃)₂ OH A B C D E F OCH₃ GH I O—CH₃COOH J K CH₃ L M N Cl O CH₂OH P Q OPO(O—CH₂—CH₃)₂ R NOH S F THCOO U V N(OCH₂CH₃)₂ W

EXAMPLES Example 1. Percentage of Total PUFAs in Membranes of CellsTreated with D-PUFAs and PUFAs

Synthetic D-PUFA molecules are hydrophobic, and therefore cells exposedto these D-PUFAs have high levels of these fatty acids on theirsurfaces.

Table 4 shows the total percentage of PUFAs in membranes of 3T3 cellstreated with 100 μM of these fatty acids for 48 hours. To perform theseexperiments, membranes were extracted and total fatty acids wereobtained by hydrolysis in basic medium. Methanolic bases of these fattyacids were quantified by gas chromatography. The data shown are averagesof four independent measures of PUFA's mass divided by the total fattyacids and expressed as a percentage. It is also shown is standard errorof the mean. In cell cultures, 3T3 cells incubated in the presence ofthese fatty acids showed higher levels of PUFAs (including D-PUFAs) andlower levels of saturated fatty acids.

The control corresponds to a culture without the presence of addednatural or synthetic fatty acids. Cells in their natural form presentPUFAs in their membranes, but the presence in the medium of the D-PUFAmolecules of the invention increases these levels of PUFAs in the cellmembrane. Therefore these results suggest that nutraceutical orpharmaceutical interventions of these compounds of the present inventioncan effectively regulate the composition of the cell membranes.

TABLE 4 Lipid added Percentage of total PUFA None (Control) 32.4 ± 2.1182A1 42.3 ± 3.1 183A1 42.8 ± 2.2 183A2 44.0 ± 2.6 204A1 45.5 ± 2.9205A1 46.7 ± 3.4 226A1 48.9 ± 3.7

Example 2. L (Lamellar)-to-H_(II) (Hexagonal) Transition in DEPE(Dielaidoil Phosphatidylethanolamine) Cell Membranes

Tables 5 and 6 show the lamellar-to-hexagonal (H_(II)) transitiontemperature in DEPE model membranes. The transition temperature wasdetermined by Differential Scanning Calorimetry. The proportionDEPE:D-PUFA was 10:1 (mol:mol) in all cases. Lamellar-to-hexagonaltransition is an important parameter that reflects relevant signallingproperties of cell membranes. The propensity to form H_(II) phases,which is higher as the temperature of this transition lowers indicatesthat the membrane surface pressure is lower, meaning that the polarheads of phospholipids form a less dense or compact network that thoseformed by lamellar structures (Escribá et al., 2008). When this occurs,certain peripheral membrane proteins (such as G proteins, protein kinaseC or Ras protein) can more easily bind to the membrane, while othershave a poor interaction (e.g., the Gα-protein), so changes in the H_(II)transition temperature are important in regulating cellular functionsrelated to health and human therapy (Escribá et al., 1995, Vogler etal., 2004; Escribá, 2006).

Control values correspond to model membranes in the absence of fattyacids. The reduction in H_(II) transition temperature obtained by usingthe D-PUFA of the invention indicates an increased induction of membranediscontinuities, generating anchoring sites in the membrane forperipheral proteins and leads to better regulation of cell signallingand, therefore, greater effectiveness in the control of certaindiseases.

Thus Table 5 shows the transition temperature T_(H) (hexagonal lamellarto H_(II)) in membranes of DEPE (4 mM) in the presence or absence of 200μM of various compounds of the present invention of the series A.

TABLE 5 Transition Lipid added temperature None (Control) 64.5 182A151.8 183A1 51.6 183A2 50.1 204A1 49.3 205A1 47.9 226A1 44.4

Table 6 shows the temperature of lamellar-to-hexagonal transition inDEPE membranes in the presence of D-PUFAs from several series.

TABLE 6 182 183-1 183-2 204 205 226 B 52.1 51.9 51.0 50.2 48.3 45.1 D51.0 51.1 49.4 48.7 47.5 43.9 E 50.6 49.8 49.3 48.4 46.7 42.9 G 51.050.3 50.1 49.6 47.3 44.1 O 51.7 51.2 51.3 49.7 48.6 44.2 R 52.2 51.849.9 50.0 48.4 44.7

Example 3. Binding of Gi₁ Protein (Trimer) to a Model Cell Membrane

The regulation of the membrane lipid composition resulted in changes inmembrane structure, as measured by Differential Scanning Calorimetry,which causes variations in the localization of G proteins in model cellmembranes as shown in Table 7. The net result is a regulation of cellsignalling leading to the reversal of various pathological processes, asshown later. Table 7 shows the binding of heterotrimeric Gi₁ protein tomodel membranes of phosphatidylcholine:phosphatidylethanolamine (6:4,mol:mol) measured by centrifuge analyses, followed by immunoblotting,visualization by chemiluminescence and quantified by image analysis. Forthese experiments it was used 2 mM phospholipid and 0.1 μM of thedifferent D-PUFAs indicated in Table 7. The Control is a sample of modelmembranes in the absence of fatty acids.

These results indicate that the modification induced in the structuraland functional properties of the membrane increases as the number ofunsaturations increases. Both the presence of unsaturations and thechanges in carbons 1 and 2 reduce the rate of metabolism of PUFAs. Thisfact, in relation with the particular effect of these lipids on themembrane structure, indicates that the action on the abnormal cellsshare a common origin.

In fact, there was a good correlation between the pharmacological effectand the effect they have on the lipid membrane structure.

TABLE 7 Lipid added G protein binding None (Control) 100 ± 5  182A1 312± 12 183A1 328 ± 9  183A2  17 ± 357 204A1 385 ± 22 205A1 406 ± 14 226A1422 ± 26

Example 4. Use of 1,2-PUFA Derivatives for the Treatment of Cancer

Cancer is a disease characterized by the uncontrolled proliferation oftransformed cells. As indicated above, in addition to certain geneticalterations, cancer is characterized by the presence of altered levelsof membrane lipids that may influence cell signalling. In this sense,the natural PUFAs showed some efficacy against the development of humancancer cells (A549) at the concentrations used in this study, althoughits metabolic use probably prevented a greater efficacy (FIG. 1).However, D-PUFAs showed a marked and significantly higher efficacy thanthe unmodified molecules at carbons 1 and 2 (FIG. 1 and Table 8) at thesame concentrations. These results indicate that the changes on naturalpolyunsaturated fatty acids results in molecules with strong anti-tumourpotency and significantly greater than that of natural PUFAs andtherefore have great utility in the treatment and prevention of tumourdiseases through pharmaceutical and nutraceutical approaches in humansand animals.

For the experiments shown in FIG. 1, cultured human Non Small Cell LungCancer cells (A549) in RPMI 1640 were used, supplemented with 10% foetalbovine serum and antibiotics, at 37° C. and 5% CO₂. Cells weremaintained in culture for 48 hours in the presence or absence of D-PUFAsand PUFAs indicated in Table 2 at a concentration of 250 μM. Aftertreatment, cell count was performed and the study of the mechanismsinvolved in the antitumor activity of compounds was evaluated by flowcytometry. FIG. 1 shows the percentage of cell survival (being assigned100% to the untreated tumour cells). These values correspond to averagesof three independent experiments.

In a separate series, compounds listed in Table 3 were used againstdifferent tumour types shown in Tables 8A, 8B and 8C. These charts showthe antitumor efficacy of the compounds of this invention against thegrowth of breast cancer cells, brain (glioma), and lung cancer. Efficacydata are expressed as IC₅₀ values (values of μM concentration whichproduce death in 50% of tumour cells) after 72 hours of incubation. Theother experimental conditions are identical to those described in thepreceding paragraph.

The results clearly indicate that all D-PUFAs are highly effectiveagainst tumour development. Overall, it may be seen that the series ofcompounds A and B are the best, so the effectiveness of these seriesagainst the development of leukaemia and liver cancer (Tables 9 and 10)was tested. Also, it can be argued that the compounds of the series 204and 226, i.e., numbered D-PUFAs with the pair number of instaurationshigher in size, are most effective. These results indicate the existenceof a structure-function relationship in the pharmacological activity ofthe present invention, which also goes in favour of the thesis of acommon mechanism of action related to the structure of each compoundand, therefore, of the unity of invention in this section.

Table 8A shows the efficacy of the compounds of the invention to controlthe growth of breast cancer cells MDA-MB-231, expressed in micromolarIC₅₀ values.

TABLE 8A Molecule Series Subseries 182 183 (1) 183 (2) 204 205 226 A 388380 347 381 390 187 B 379 267 156 345 208 195 C 386 289 168 389 223 210D 277 245 175 281 2 224 E 289 319 193 299 284 207 F 311 323 181 326 275226 G 378 364 159 372 219 213 H 402 308 170 363 282 199 I 411 274 210315 261 241 J 287 296 221 285 228 235 K 375 381 238 317 240 208 L 343306 173 332 253 216 M 362 407 164 321 216 267 N 297 278 186 274 289 222O 286 267 217 298 264 249 P 419 349 214 370 301 250 Q 328 312 205 306247 263 R 371 305 172 285 245 204 S 388 291 189 293 270 211 T 391 290216 317 233 199 U 410 344 228 369 272 227 V 442 326 241 352 298 215 W391 311 203 311 256 246

Table 8B shows the efficacy of the compounds of the invention againstbrain cancer cell growth (glioma) U118, expressed in micromolar IC₅₀values.

TABLE 8B Molecule Series Subseries 182 183 (1) 183 (2) 204 205 226 A 197397 372 197 400 214 B 198 202 377 396 391 196 C 208 na 379 287 442 237 D221 na 385 311 467 241 E 213 na na 224 513 265 F 236 354 401 275 498 261G 205 329 394 342 426 278 H 267 408 443 263 439 294 I 240 321 432 328510 327 J 254 296 426 296 487 283 K 221 257 418 380 474 272 L 229 231460 247 435 269 M 238 349 407 309 462 306 N 247 324 385 315 513 285 O na370 na na na 277 P na 285 389 291 432 290 Q na 282 392 324 419 254 R 255307 454 501 468 267 S 203 316 416 462 475 315 T 214 368 423 385 427 263U 212 343 380 263 454 342 V 231 274 402 345 510 269 W 246 na 438 287 443318

Table 8C shows the efficacy of the compounds of the invention againstthe growth of lung cancer cells A549, expressed in micromolar IC₅₀values.

TABLE 8C Molecule Series Subseries 182 183 (1) 183 (2) 204 205 226 A 944200 192 243 394 195 B 196 195 197 413 202 198 C 635 281 241 521 325 214D 541 326 267 372 364 221 E 387 294 243 475 413 209 F 354 347 259 392338 286 G 439 273 295 427 407 273 H 462 319 219 398 290 247 I 673 348276 459 351 298 J 321 281 259 362 416 215 K 274 276 2 414 275 250 L 385285 283 326 362 221 M 286 322 248 375 293 208 N 329 379 255 420 384 236O 452 344 318 461 418 264 P 328 317 272 387 339 291 Q 293 273 314 348365 252 R 317 258 274 364 417 219 S 458 341 246 439 293 265 T 379 367279 352 322 243 U 255 294 287 270 426 270 V 340 320 291 326 325 298 W416 352 212 341 420 302

Table 9 shows the efficacy of the compounds of the invention against thedevelopment of human leukaemia (Jurkat cells) Values of IC₅₀ micromolarat 72 hours.

TABLE 9 Molecule Series Subseries 182 183 (1) 183 (2) 204 205 226 A 713198 184 62 376 85 B 377 196 184 104 294 175

Table 10 shows the efficacy of the compounds of the invention againstthe development of liver cancer (HepG2 cells). Values of IC₅₀ micromolarat 72 hours.

TABLE 10 Compound 182 183 (1) 183 (2) 204 205 226 A 212 380 380 192 401164

All these results indicate that the D-PUFAs are useful for theprevention and treatment of cancer included in nutraceutical andpharmaceutical compositions in humans and animals. It was also foundthat the potency of action of D-PUFA is correlated with the increasednumber of double bonds and that the presence of changes in carbon 1 and2 is essential for the antitumor potency of the lipids to be relevant attherapeutic level. Because these compounds have anti-tumour effectagainst a wide range of tumour cells, it may be affirmed that they aremolecules with broad anti-tumour spectrum and may be of generalapplication against the development of any cancer.

Example 5. Use of 1,2-PUFA Derivatives for the Treatment ofCardiovascular Disease

To investigate the usefulness of the D-PUFA for the treatment ofcardiovascular diseases, several experimental approaches were used.First, the efficacy of the compounds of the invention in aorta cells inculture (cell line A-10) was investigated. These cells were maintainedin culture with complete medium (C, supplemented with 10% foetal bovineserum and PDGF) and incomplete medium (CSS, supplemented with 1% foetalbovine serum without PDGF). Cultures were performed for a period of 72hours in a similar fashion as described in the preceding paragraph.After this period of incubation, cell counts were carried out by flowcytometry.

In the incomplete medium (CSS, no extra control PDGF), cells have aproliferative behaviour, similar to that produced in a healthy body. Theproliferative behaviour that occurs in complete medium would be asimilar situation to what occurs in a pathological organism. Thepresence of D-PUFA produced a significant reduction in the proliferationof normal aorta (A-10) cells in complete culture medium withproliferative agents present in the foetal serum included in the culturemedium. In the presence of proliferative agents (cytokines, growthfactors, etc.), A10 cell counts were similar to those obtained inincomplete medium (CSS) with the presence of the D-PUFA of the presentinvention (FIG. 2). In contrast, PUFA showed little or noantiproliferative efficacy, demonstrating that the changes made on thesefatty acids increase substantially their pharmacological potential fortreating cardiovascular diseases such as hypertension, atherosclerosis,ischemia, cardiomyopathies, aneurysms, ictus, angiogenesis, cardiachyperplasia, infarction, angina, stroke (cerebrovascular accidents),etc.

The effects on this cell line can not be considered toxic for tworeasons: (1) in complete medium, D-PUFAs never induced reductions incell proliferation below the levels of cells incubated in incompletemedium, and (2) aorta (A10) cells treated with D-PUFAs showed no signsof molecular or cellular necrosis, apoptosis or any other type of celldeath. Since the proliferation of vascular cells is involved in thedevelopment of numerous cardiovascular diseases, D-PUFAs are useful forthe prevention and treatment of these diseases through nutraceutical andpharmaceutical approaches in humans and animals.

In a separate series, rat cardiomyocytes were isolated and cultured invitro for 24 hours, after which a number of parameters were measured.First, it was measured the number, length and width of cells in culture.It was observed that all compounds of series A and B (182-226) were ableto increase the number of cells that survived in culture (between 12%and 33%) and their length and width (between 18% and 42%). In addition,these compounds induce decreases in the release of lactate dehydrogenase(LDH) induced by anoxia (reductions of between 9% and 68% for allcompounds of series A and B). These results indicate that the D-PUFAmolecules of the present invention have a protective effect oncardiovascular cells and increase their elasticity, which can be used toprevent and treat heart and vascular diseases of various kinds, such ashypertension, atherosclerosis, ischemia, cardiomyopathy, aneurysm,ictus, angiogenesis, cardiac hyperplasia, infarction, angina, stroke(cerebrovascular accident), faulty blood circulation, etc.

In a separate experimental series, it was studied the effect of D-PUFAmolecules of the present invention on blood pressure of SHR rats. Inthese animals both, blood pressure and levels of apolipoprotein AI(apoA-I) were measured. For these experiments Spontaneously HypertensiveRats (SHR) were treated for 30 days with vehicle (water control) orcompounds of the invention (200 mg/kg day, p.o.). At the end of thisperiod, the animals' blood pressure and serum levels of apoA-I weremeasured. The results show the capacity of the compounds of the presentinvention to lower blood pressure and induce the expression of apoA-I,indicating that these molecules are useful in the treatment ofhypertension and atherosclerosis (Table 11). For these experiments,non-invasive methods for determining blood pressure (cuff-tail method)and gene expression for apoA-I (RT-PCR) described in the literature(Terés et al., 2008) were used. The usefulness of the molecules of thepresent invention for the treatment of cardiovascular diseases isreinforced by its capacity for reducing the levels of serum cholesteroland triglycerides (see below).

Table 11 shows the blood pressure (mm Hg) and levels of apoA-I (%) inSHR rats. The average values of SHR before treatment were 214 mmHg and100% respectively.

TABLE 11 Compound 182 183 (1) 183 (2) 204 205 226 A 204 201 189 205 193194 146 134 311 131 346 324 B 201 197 182 202 187 186 178 151 285 144264 333 F 198 203 191 199 195 202 192 146 279 163 319 357 L 207 205 194197 198 200 131 125 268 188 376 296 N 187 208 194 201 189 199 159 189296 174 293 348 P 202 201 187 203 194 193 184 178 347 153 337 382 V 207199 198 198 191 195 166 152 282 161 315 324

Example 6. Use of 1,2-PUFA Derivatives for the Treatment of Obesity

FIG. 3A shows how PUFAs (both natural and synthetic ones) are capable ofinhibiting the hyperplasia and hypertrophy of fat cells. For this study,3T3-L1 adipocytes were used. This effect was already known and had beendescribed previously for unmodified natural PUFAs (Hill et al., 1993).However, D-PUFAs have an increased potency to inhibit the proliferationof fat cells (FIG. 3A). This effect is not toxic in any case, sinceinhibition of growth of fat cells did not produce reductions in cellproliferation below levels of cells cultured in incomplete medium (with1% serum). The cell culture media and conditions used were similar tothose described above.

These results demonstrate that D-PUFAs have a high potential to inhibitthe growth of fat cells and, therefore, for the prevention and treatmentof obesity and other processes related to the accumulation of bodyadipocytes (e.g., cellulite) or appetite alterations throughnutraceutical or pharmaceutical approaches in animals and humans. Theobserved effect, again, showed a clear correlation with the number ofdouble bonds of the molecules used and the presence of modifications atcarbons 1 and 2 in the lipid molecule.

Additionally, several compounds related to the present invention wereused to study their effect on body weight of rats (FIG. 3B). In thisregard, Spontaneously Hypertensive Rats (SHR) treated with compounds182-226 (series A, B, N and P) showed reductions in body weight after 1month treatment with 200 mg/kg (reductions of 3.2% to 6.9%) caused inpart by a decrease in food intake and partly by inhibition of theproliferation of fat cells (in untreated animals fed with the sameamount of food the weight drop was not as marked as in animals treated).These results demonstrate that these compounds can be used in thecontrol of body weight (obesity and overweight), appetite control andbody fat (cellulite) regulation.

Example 7. Use of 1,2-PUFA Derivatives for the Treatment ofNeurodegenerative Diseases

In these studies, different models of neurodegeneration were used.First, P19 cells were studied, where neuronal differentiation wasinduced with trans-retinoic acid. To do this, P19 cells were incubatedin minimum essential medium (α-MEM) supplemented with 10% foetal bovineserum and 2 μM of trans retinoic acid at 37° C. in the presence of 5%CO₂. Cells were incubated in the presence or absence of several D-PUFAsor PUFAs at different concentrations for 24 hours. Neurotoxicity wasinduced with 1 μM NMDA. Subsequently, the number of cells was counted byoptical microscopy in the presence of trypan blue. These experimentsshowed that PUFAs have a protective effect on neuronal degeneration,although the effect mediated by D-PUFAs is much higher (FIG. 4A andTable 12). In this figure and table it is clear that the D-PUFAmolecules of the present invention protect against neuronal death, asthey inhibit NMDA-induced neuronal death, so that these substances maybe useful for the prevention and treatment of neurodegenerative diseasessuch as Alzheimer's disease, sclerosis, Parkinson's disease,leukodystrophy, etc. It has also been shown that the number of cells incultures treated is higher than in cultures were there are notneurodegenerative agents added. In particular, cell death negativevalues indicate that the number of P19 cells is higher than in a controlsituation. Therefore, the D-PUFA compounds of the present invention canbe used to promote neuroregenerative processes, such as those producedby traumatic processes (accident) or toxic agents.

Table 12 shows the protective effect against neuronal death in P19cells: inhibition of neuronal death (P19 cells) with D-PUFA of thepresent invention after treatment with NMDA (100% death). Control cellswithout NMDA, showed a level of 0% cell death. All percentages below100% indicate protection against neuronal death. Negative valuesindicate that in addition to protection of neuronal death there is alsoa level of neuronal proliferation. Furthermore, the compounds of thepresent invention decrease the levels of α-synuclein (Table 13), aprotein that is associated with neurodegenerative processes, such asParkinson's, Alzheimer's, dementia of Lewy, multiple systemic atrophy,prion diseases, etc. Therefore, the molecules of the present inventioncan be applied to the prevention and treatment of neurodegenerative,neuroregenerative, neurological and neuropsychiatric processes.

TABLE 12 182 183-1 183-2 204 205 226 C (NMDA) A −60 −55 −70 −70 −50 −230100 B −62 −58 −66 −71 −52 −222 100 F −45 −35 −36 −46 −44 −189 100 L −32−21 −29 −27 −35 −117 100 V −17 −9 −18 −11 −27 −86 100

Table 13 shows the expression of α-synuclein in neuronal cultures (cellsP19). C (control) represents the % of α-synuclein in untreated cells(100%).

TABLE 13 182 183-1 183-2 204 205 226 C A 50 45 40 41 35 23 100 B 61 4338 36 41 31 F 71 61 52 52 57 41 L 80 76 73 69 67 64 V 83 87 89 82 81 77

To test the efficacy of the compounds of the present invention to induceneuroregeneration or inhibit neurodegeneration, an animal model ofAlzheimer's disease was used. In this model mice developneurodegeneration because they express a series of mutant proteins thatlead to brain damage (Alzh mice). B6 mice were used as healthy animalcontrols. Both groups of animals were treated for a period of 3 monthswith vehicle (water) or with various D-PUFA (20 mg/kg, daily po) sincethey were an age of 3 months. To determine whether cognitive improvementoccurred after treatment, animal behaviour was monitored in the radialmaze. The animals are kept on restricted diet to have appetite. In asymmetrical 8-arm radial maze, visual marks were placed to facilitatethe orientation of the animal and food (15 mg tablet) was put in four ofthe arms. The time each animal took to complete the exercise, and thenumber of errors, were measured using a camera attached to a computersystem. In this sense, Alzheimer animals have values about 50% higherthan healthy animals, both by the time it takes to perform the exerciseand by the number of errors made (FIG. 4B). By contrast, mice withAlzheimer treated with 226B1 (Alzh+LP226) presented behaviouralparameters similar to those of control animals and significantly(P<0.05) lower than animals treated with vehicle (Alzh). In this regard,the effectiveness of the compound 183B1, 205A1, 205B1, 226A1, 226 V1 wasalso tested, showing improvements in animals with Alzheimer's disease(times of 98, 92, 93, 86 and 89 seconds, respectively). On the otherhand, it is also interesting that these same compounds (183B1, 205A1,205B1, 226A1, 226B1 and 226V1) also produced reductions in the timestaken to complete the experiment in control animals (B6 healthy mice) of8 s, 11 s, 12 s, 18 s, 16 s and 14 s, respectively. Therefore, it may beconcluded that these compounds have significant activity againstneurodegeneration and in neuroregeneration. Among the neurodegenerativeprocesses that could be prevented and treated with D-PUFA molecules ofthe present invention are Alzheimer's disease, Parkinson disease,Zellweger syndrome, multiple sclerosis, amyotrophic lateral sclerosis,the sclerosis of the hippocampus and other types of epilepsy, focalsclerosis, adrenoleukodystrophy and other types of leukodystrophy,vascular dementia, senile dementia, dementia of Lewy, multiple systemicatrophy, prion diseases, etc. In addition, neuroregenerative activity,evidenced by the effect in both mice with Alzheimer and healthy B6 mice,treatment can be applied to processes in which neuronal loss hasoccurred as a result of an accident, surgery, trauma of different natureor due to certain toxins. D-PUFA molecules of the present invention canalso be used for the prevention or treatment of different neurologicaland/or neuropsychiatric problems, such as headaches including migraine,central nervous system injury, sleep disorders, dizziness, pain, stroke(cerebrovascular accidents), depression, anxiety, addictions, memory,learning or cognitive problems, and for enhancing the memory andcognitive ability of human beings.

Example 8. Use of 1,2-PUFA Derivatives for the Treatment of InflammatoryDiseases

Cyclooxygenase (COX) is an enzyme that can bind to membranes, takingcertain lipids from there and catalyze its conversion into moleculesthat can have inflammatory activity. The binding of this enzyme tomembrane lipids is due in part to the membrane lipid structure. Theincreased activity of COX 1 and 2 isoforms has been associated with theetiopathology of a number of inflammatory diseases by inhibitingarachidonic acid metabolism to produce pro-inflammatory lipid mediators.The D-PUFA compounds of the present invention produced a series ofcellular signals that alter the metabolism of arachidonic acid and, as aresult, they inhibit the activity and expression of COX in monocytes inculture (Table 14 and FIG. 5A). Also, the D-PUFA of the presentinvention inhibited the production of pro-inflammatory cytokines (TNF-α)in vivo (Table 15 and FIG. 5B). For this purpose, C57BL6/J mice weretreated with the various derivatives (200 mg/kg, p.o.) after inducing aninflammatory reaction in them by intraperitoneal injection of 20 μg ofbacterial lipopolysaccharide (LPS). These results clearly indicate theeffectiveness of the D-PUFA of the present invention to prevent orreverse inflammatory processes and pathologies.

Table 14 shows the expression of COX-2 in monocytes in culture.Inhibition of COX-2 expression in monocytes. Percentages of inhibition(compared to the positive control in the presence of LPS, 100%) of COX-2protein levels (expression) by the various fatty acid derivatives.

TABLE 14 182 183-1 183-2 204 205 226 C (LPS) A 24 20 23 17 31 23 100 B39 33 29 28 39 37 F 56 46 36 41 47 49 L 67 65 48 47 53 69 V 81 79 68 4376 85

Table 15 shows the production of TNF-α (%) in mice: percentage of TNF-αin serum after injection of LPS (20 μg) intraperitoneally in C57BL6/Jmice (100%).

TABLE 15 182 183-1 183-2 204 205 226 C (LPS) A 64 70 71 24 56 73 100 B79 81 78 26 69 83 F 86 91 86 46 80 91 L 85 86 91 49 76 88 V 81 84 87 4284 85

These results show that the molecules of the present invention can beuseful for preventing or treating inflammatory diseases, includinginflammation, cardiovascular inflammation, inflammation caused bytumours, inflammation of rheumatoid origin, inflammation caused byinfection, respiratory inflammation, acute and chronic inflammation,hyperalgesia of inflammatory nature, oedema, inflammation resulting fromtrauma or burns, etc.

Example 9. Use of 1,2-PUFA Derivatives for the Treatment of MetabolicDiseases

Lipids are critical molecules in maintaining metabolic functions. PUFAtreatments produced some modest reductions in cholesterol andtriglycerides levels in 3T3-L1 cells. However, D-PUFA treatmentsresulted in marked and significant reductions in cholesterol andtriglyceride levels in these cells. For these experiments, the abovementioned cells were incubated in RPMI 1640 medium in presence of 10%foetal bovine serum at 37° C. with 5% CO₂ and in the presence or absenceof 150 μM of different PUFA or D-PUFA. The cells were incubated for 24 hand then subjected to lipid extraction and cholesterol and triglyceridelevels were measured following the procedures described previously(Folch et al., 1951).

In a separate experimental series, SHR rats were treated with variouscompounds of the present invention (200 mg/kg daily, 28 days, p.o.) andthe levels of cholesterol, triglycerides and glucose in serum weremeasured by colorimetric methods. It was observed that these compoundsinduce significant (and in many cases marked) reductions in the levelsof these metabolites (Table 16).

The results shown in FIGS. 6A and 6B and Table 16 clearly indicate thatthe D-PUFAs can be used as drugs for the treatment or prevention ofmetabolic diseases, such as hypercholesterolemia, hypertriglyceridemia,diabetes and insulin resistance in humans and animals, throughpharmaceutical and nutraceutical approaches. The combination high levelsof cholesterol and triglycerides, high glucose, together withcardiovascular and/or body weight alterations leads to “metabolicsyndrome”, which is beginning to increase in Western societies. Thecompounds of the present invention have great therapeutic potential fortreating metabolic syndrome.

Table 16 shows the levels of cholesterol, triglycerides and glucose inSHR rats. It shows the value of cholesterol (top number), triglycerides(central number) and glucose (bottom number) in serum of SHR treatedwith the molecules described above (200 mg/kg daily, p.o., 28 days).Values are expressed as percent, and in untreated (control) rats valueswere always considered as 100%.

TABLE 16 Compound 182 183 (1) 183 (2) 204 205 226 A 78 76 79 72 69 64 9181 78 77 74 71 84 87 82 85 82 79 B 89 75 77 71 58 59 72 66 76 69 65 6287 84 86 89 87 81 F 92 78 84 76 71 67 88 71 87 81 83 78 89 76 85 84 8286 L 89 82 83 83 79 71 93 77 79 82 78 74 94 85 92 91 85 87 N 92 72 89 8280 75 93 69 85 81 73 72 90 84 92 82 86 83 V 94 75 84 84 85 81 93 70 9281 79 84 93 79 88 87 84 89

Example 10. Structural Basis of the Therapeutic Effects of1,2-Derivatives of PUFAs

Numerous studies have shown that the intake or treatment with lipidresults in changes in the lipid composition of cell membranes.Furthermore, such composition has a direct effect on the membrane lipidstructure, which in turn regulates cell signalling and is related to theoccurrence of many diseases. FIGS. 7A and 7B show the correlationbetween changes in the structure of the membrane produced by differentD-PUFAs (as measured by the H_(II) transition temperature) and thecellular effects observed in this study. For this purpose, we determinedthe mean effect of each of the D-PUFAs (average of each lipid for alldiseases studied with respect to the number of double bonds) and theresults have been plotted against the transition temperature. Thereduction in H_(II) transition temperature indicates a greater inductionof discontinuities in membranes, creating docking sites for peripheralmembrane proteins that leads to a better regulation of cell signalling,and therefore a more effective control of certain diseases. To someextent, the fact that complex organisms can metabolize drugs and thatsome additional mechanisms may be operating in some types (subtypes) ofdiseases, suggests that some of the molecules with fewer double bondscan have greater pharmacological activity. However, in general, itappears that the therapeutic effect depends on the number of doublebonds of the molecule, which itself is related to the capacity ofregulating the structure of the membranes. In that sense, the presenceof radicals in carbons 1 and/or 2, found in the D-PUFA compounds of thepresent invention, but not in natural PUFAs, is essential to enhance thetherapeutic effect of these molecules.

These results indicate that the effects of lipids contained in thisinvention have a common basis. These correlations (with r² values of0.77 and 0.9 for D-PUFAs and P<0.05 in both cases) clearly indicate thatthe structure of the lipids used is the basis of its effect and that itoccurs through the regulation of membrane structure, caused by thestructure-function relationship of each lipid.

Thus, the present invention relates in a first aspect to compounds offormula (I) or pharmaceutically acceptable derivatives where a, b and cindependently can have values from 0 to 7 and R₁ and R₂ may be an ion,atom or group of atoms with a molecular weight not exceeding 200 Daindependently, for use in the treatment of diseases based on structuralalterations and/or functional characteristics of cell membrane lipidsselected from: cancer, vascular disease, inflammation, metabolicdiseases, obesity, neurodegenerative diseases and neurologicaldisorders.

A second aspect of the present invention relates to the use of at leastone compound of formula (I), or its pharmaceutically acceptablederivatives, where a, b and c independently may have values from 0 to 7,and R₁ and R₂ can be an ion, atom or group of atoms with a molecularweight not exceeding 200 Da independently, for the preparation of apharmaceutical and/or nutraceutical composition for the treatment ofdiseases based on structural and/or functional alterations of lipids incell membranes selected from: cancer, vascular diseases, inflammation,metabolic diseases, obesity, neurodegenerative diseases and neurologicaldisorders.

The last aspect of the present invention relates to a method fortherapeutic treatment of diseases in humans and animals whose commonetiology is related to structural and/or functional alterations of thelipids located in cell membranes selected from: cancer, vasculardisease, inflammation, metabolic diseases, obesity, neurodegenerativeand neurological diseases, which comprises administration to the patientof a therapeutically effective amount of at least one compound offormula (I) and/or its pharmaceutically acceptable salts or derivatives,where a, b and c can have independent values between 0 and 7, and R₁ andR₂ may be an ion, atom or group of atoms with a molecular weightindependently not exceeding 200 Da.

REFERENCES

-   Alemany et al. 2004. Hypertension, 43 249-   Alemany et al. 2007. Biochim Biophys Acta, 1768, 964-   Buda et al. 1994. Proc Natl Acad Sci U.S. A., 91, 8234-   Coles et al. 2001. J Biol Chem, 277, 6344-   Escribá et al. 1995. Proc Natl Acad Sci U.S. A., 92, 7595-   Mail et al. 1997. Proc. Natl. Acad. Sci USA., 94, 11 375-   Escriba et al 2003. Hypertension, 41, 176-   Escriba 2006. Trends Mol Med, 12, 34-   Escriba et al. in 2008. J Cell Mol Med, 12, 829-   Florent et al. 2006. J Neurochem., 96, 385-   Folch et al. 1951. J Biol Chem, 191.83-   Jackson and Schwartz 1992. Hypertension, 20, 713-   Jung et al. in 2008. Am J Clin Nutr 87, 2003S-   Lane and Farlow 2005. J Lipid Res, 46, 949-   Martinez et al. 2005. Mol Pharmacol., 67, 531-   Rapoport 2008. Postraglandins Leukot. Essent. Fatty Acids 79,    153-156-   Sagin and Sozmen 2008. J Lipid Res, 46, 949-   Schwartz et al. 1986. Circ Res 58, 427-   Stender and Dyerberg 2004. Ann Nutr Metab., 48, 61-   Terés et al., 2008. Proc. Natl. Acad. Sci USA, 105, 13 811-   Trombetta et al. 2007. Chem Biol Interact., 165, 239-   Vogler et al 2004. J. Biol Chem, 279, 36 540-   Vogler et al 2008. Biochim Biophys Act, 1778, 1640-   Yang et al. 2005. Mol Pharmacol., 68, 210

1-16. (canceled)
 17. A method to treat a cancer in a subject in needthereof comprising administering to the subject an effective amount of acomposition comprising at least a polyunsaturated fatty acid selectedfrom the group consisting of (i)COOH—CHOH—(CH₂)₆—(CH═CH—CH₂)₂—(CH₂)₃—CH₃ (182A1), an acceptable salt, ora combination thereof; (ii) COOH—CHOH—(CH₂)₆—(CH═CH—CH₂)₃—CH₃ (183A1),an acceptable salt, or a combination thereof; (iii)COOH—CHOH—(CH₂)₃—(CH═CH—CH₂)₃—(CH₂)₃—CH₃ (183A2), an acceptable salt, ora combination thereof; (iv) COOH—CHOH—(CH₂)₂—(CH═CH—CH₂)₄—(CH₂)₃—CH₃(204A1), an acceptable salt, or a combination thereof; (v)COOH—CHOH—(CH₂)₂—(CH═CH—CH₂)₅—CH₃ (205A1), an acceptable salt, or acombination thereof; (vi) COOH—CHOH—CH₂—(CH═CH—CH₂)₆—CH₃ (226A1), anacceptable salt, or a combination thereof; or, (vii) a combinationthereof.
 18. A method to treat a neurodegenerative disease in a subjectin need thereof comprising administering to the subject an effectiveamount of a composition comprising at least a polyunsaturated fatty acidselected from the group consisting of (i)COOH—CHOH—(CH₂)₆—(CH═CH—CH₂)₂—(CH₂)₃—CH₃ (182A1), an acceptable salt, ora combination thereof; (ii) COOH—CHOH—(CH₂)₆—(CH═CH—CH₂)₃—CH₃ (183A1),an acceptable salt, or a combination thereof; (iii)COOH—CHOH—(CH₂)₃—(CH═CH—CH₂)₃—(CH₂)₃—CH₃ (183A2), an acceptable salt, ora combination thereof; (iv) COOH—CHOH—(CH₂)₂—(CH═CH—CH₂)₄—(CH₂)₃—CH₃(204A1), an acceptable salt, or a combination thereof; (v)COOH—CHOH—(CH₂)₂—(CH═CH—CH₂)₅—CH₃ (205A1), an acceptable salt, or acombination thereof; (vi) COOH—CHOH—CH₂—(CH═CH—CH₂)₆—CH₃ (226A1), anacceptable salt, or a combination thereof; or, (vii) a combinationthereof.